Knockdown of desmin in zebrafish larvae affects interfilament spacing and mechanical properties of skeletal muscle

Abstract

Skeletal muscle was examined in zebrafish larvae in order to address questions related to the function of the intermediate filament protein desmin and its role in the pathogenesis of human desminopathy. A novel approach including mechanical and structural studies of 4-6-d-old larvae was applied. Morpholino antisense oligonucleotides were used to knock down desmin. Expression was assessed using messenger RNA and protein analyses. Histology and synchrotron light-based small angle x-ray diffraction were applied. Functional properties were analyzed with in vivo studies of swimming behavior and with in vitro mechanical examinations of muscle. The two desmin genes normally expressed in zebrafish could be knocked down by 50%. This resulted in a phenotype with disorganized muscles with altered attachments to the myosepta. The knockdown larvae were smaller and had diminished swimming activity. Active tension was lowered and muscles were less vulnerable to acute stretch-induced injury. X-ray diffraction revealed wider interfilament spacing. In conclusion, desmin intermediate filaments are required for normal active force generation and affect vulnerability during eccentric work. This is related to the role of desmin in anchoring sarcomeres for optimal force transmission. The results also show that a partial lack of desmin, without protein aggregates, is sufficient to cause muscle pathology resembling that in human desminopathy.

POMKmutation in a family with congenital muscular dystrophy with merosin deficiency, hypomyelination, mild hearing deficit and intellectual disability

“…In cardiomyocytes of Large knockout mice ( Large myd ), the deficiency of α-dystroglycan glycosylation not so much impaired its adhesion to laminin but more the sarcolemmal integrity of the myocytes during cyclical cell stretching with influx of membrane-impermeant dye 42. On the other hand, morpholino-mediated knockdown of desmin by ≈50% in zebrafish lowered tension of the myofibrils but made them more resistant against acute eccentric stretch-induced injury 43. It is therefore difficult to fathom whether the secondary desmin deficiency in our patient would aggravate or ameliorate their muscle dystrophy.…”

Muscle dysfunction and structural defects of dystrophin‐nullsapjemutant zebrafish larvae are rescued by ataluren treatment

“…The larvae preparations (5 dpf) were mounted, as described previously, using aluminum clips, between a force transducer and a fixed hook (23, 24). The bath was perfused at 22°C with a 3‐morpholinopropanesulfonic acid (MOPS) buffered (pH 7.4) solution (containing in mM: 118 NaCl, 5 KCl, 1.2 MgCl 2 , 1.2 Na 2 HPO 4 , 24 MOPS, 10 glucose, and 1.6 CaCl 2 ).…”

“…Larvae were fixed at 5 dpf using 4% paraformaldehyde in phosphate‐buffered saline at 4°C overnight, and after washing and permeabilization, they were subsequently analyzed as whole‐mount preparations using a confocal microscope (LSM 510; Carl Zeiss, Oberkochen, Germany). General morphology was examined using rhodamine phalloidin staining of actin as described previously (24). Immunohistochemistry was performed using a mouse anti‐dystrophin primary antibody [MANDRA1; dilution 1:10, 2 d incubation; Developmental Studies Hybridoma Bank (DSHB), Iowa City, IA, USA) previously used for detection in zebrafish larvae (13), and an Alexa Fluor‐488‐conjugated fluorescent secondary antibody (dilution 1:200; Invitrogen, Carlsbad, CA, USA).…”

“…The larvae are transparent, genetically characterized and have a large proportion of striated muscles (21, 22). We have shown that high‐resolution data on muscle structure and function can be obtained from zebrafish larvae (23) and have recently applied this approach to study a desmin myopathy (24).…”

Developmental Alterations in Heart Biomechanics and Skeletal Muscle Function in Desmin Mutants Suggest an Early Pathological Root for Desminopathies

“…So far, desmin aggregation was observed in mouse cardiomyocytes carrying a specific desmin mutation (Wang et al, 2001a) or CRYAB mutation (Wang et al, 2001b). Similarly, morpholino (MO)-mediated knockdown in zebrafish leads to cardiac and skeletal muscle phenotype (Li et al, 2013;Vogel et al, 2009). Yet, the role of desmin in heart function is still not well known, but it has been suggested that desmin impacts the mechanical properties of the cardiomyocytes (Hnia et al, 2014).…”